A robust luminescent assay for screening alkyladenine DNA glycosylase inhibitors to overcome DNA repair and temozolomide drug resistance / 药物分析学报

Temozolomide(TMZ)is an anticancer agent used to treat glioblastoma,typically following radiation therapy and/or surgical resection.However,despite its effectiveness,at least 50％of patients do not respond to TMZ,which is associated with repair and/or tolerance of TMZ-induced DNA lesions.Studies have demonstrated that alkyladenine DNA glycosylase(AAG),an enzyme that triggers the base excision repair(BER)pathway by excising TMZ-induced N3-methyladenine(3meA)and N7-methylguanine le-sions,is overexpressed in glioblastoma tissues compared to normal tissues.Therefore,it is essential to develop a rapid and efficient screening method for AAG inhibitors to overcome TMZ resistance in glio-blastomas.Herein,we report a robust time-resolved photoluminescence platform for identifying AAG inhibitors with improved sensitivity compared to conventional steady-state spectroscopic methods.As a proof-of-concept,this assay was used to screen 1440 food and drug administration-approved drugs against AAG,resulting in the repurposing of sunitinib as a potential AAG inhibitor.Sunitinib restored glioblastoma(GBM)cancer cell sensitivity to TMZ,inhibited GBM cell proliferation and stem cell char-acteristics,and induced GBM cell cycle arrest.Overall,this strategy offers a new method for the rapid identification of small-molecule inhibitors of BER enzyme activities that can prevent false negatives due to a fluorescent background.

MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B

Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and β-myosin heavy chain (β-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3' untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.

Role of 3-methyladenine in uric acid-induced apoptosis of renal tubular epithelial cells / 中华肾脏病杂志

Objective:To investigate the effect of autophagy inhibitor 3-methyladenine (3-MA) on uric acid (UA)-induced apoptosis of renal tubular epithelial cells.Methods:(1) Totally 24 SD rats were randomly divided into 4 groups: control group, treatment with 3-MA group, hyperuricemic nephropathy (HN) group, and HN+3-MA group, with 6 rats in each group. According to the body weight of the rats, adenine (100 mg/kg) and potassium oxonate (1 500 mg/kg) were mixed with distilled water to make a suspension, and the rats were given intragastrically once daily for consecutive 21 days to establish HN rat model. The control group and the 3-MA treatment group were fed an equivalent amount of distilled water. At the same time, the 3-MA treatment group and the HN+3-MA group were intraperitoneally injected with 3-MA (15 mg/kg), and the control group and HN group were intraperitoneally injected with an equal volume of saline once daily for 21 consecutive days. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to observe renal cell apoptosis. Western blotting was used to detect the expression levels of cleaved caspase-3 and Bax, and immunofluorescence staining was used to detect the expression and localization of cleaved caspase-3 in renal tissue. (2) Human renal tubular epithelial cells (HK-2) were stimulated with UA (800 μmol/L), and cells were administrated with different concentrations of 3-MA or Beclin-1 small interfering RNA (siRNA). The apoptosis of renal tubular epithelial cells was detected by Western blotting and immunofluorescence staining.Results:Compared with the normal rats, the apoptosis of renal tubular epithelial cells in the HN group was significantly increased ( P<0.01), and the expression levels of cleaved caspase-3 and Bax were significantly up-regulated (both P<0.05). Compared with the HN group, the apoptosis of renal tubular epithelial cells in the HN+3-MA group was significantly decreased ( P<0.01). In addition, high level of uric acid could significantly increase the levels of apoptosis associated proteins in HK-2 cells (all P<0.05), and using different concentrations of 3-MA or transfecting with Beclin-1 siRNA could significantly reduce the expression of cleaved caspase-3 and Bax (all P<0.05). Conclusion:Autophagy plays an important role in uric acid-induced apoptosis of renal tubular epithelial cells. Inhibiting the excessive activation of autophagy may be a new strategy to prevent the progression of HN.

The role of autophagy in pulmonary inflammatory response in silicosis model rats / 中国职业医学

@#Objective To evaluate the role of autophagy in inflammatory response in the lungs of silicosis rats. Methods The specific pathogen-free healthy male Wistar rats were randomly divided into four groups with 10 rats in each group. The rats in the control group were given an equal volume of 0.9% sodium chloride solution; all rats in the silicon dioxide (SiO2 ) group, the SiO2 + 3-methyladenine (3-MA) group, and the SiO2 +rapamycin (RAPA) group were given a mass concentration of 0.05 mg/L of SiO2 suspension (1.0 mL/rat) to establish a rat model of silicosis using non-exposed tracheal instillation method. Two days before SiO2 exposure, the rats in SiO2 +3-MA group were intraperitoneally injected with 3-MA at a dose of 2.5 mg/kg body weight. The rats in the SiO2 +RAPA group were intraperitoneally injected with a dose of RAPA 1.0 mg/kg body weight, once a day, and once every other day after modeling, for a total of 10 injections. The pathological changes in the lung tissue of the rats in each group were evaluated using hematoxylin-eosin and Masson staining. The relative expression levels of transforming growth factor-β (TGF-β) , interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the lung tissue of the rats in each group were detected using enzyme-linked immunosorbent assay. The relative expression levels of microtubule-associated protein 1 light chain 3 (LC3) and yeast autophagy-related gene 6 (Beclin1) in lung tissue of rats in each group were detected by Western blotting. Results The pathological observation of rat lung tissue showed that scattered inflammatory nodules and interstitial inflammatory cells were found in the lung tissue of the rats in the SiO2 group. In the SiO2 +3-MA group, the inflammation in the lung tissue was more severe and the alveolar cavity had viscous secretions. The rats in the SiO2 +RAPA group had less inflammation and smaller inflammatory nodules than the SiO2 group. Compared with the control group, the TNF-α, IL-1β, TGF-β, Beclin1 protein relative expression levels and LC3 Ⅱ/Ⅰ ratio in the lung tissue were increased in the SiO2 group (all P<0.05). Compared with the SiO2 group, the relative expression levels of TGF-β and TNF-α in the lung tissue of the rats increased (all P<0.05), and the ratio of LC3 Ⅱ/Ⅰ and Beclin1 protein relative expression decreased in the SiO2+3-MA group (all P<0.05). Compared with the SiO2+ 3-MA group, the relative expression levels of TNF- α, IL-1β and TGF- β in the lung tissue of the SiO2 +RAPA group were decreased (all P<0.05), and the LC3Ⅱ/Ⅰ ratio and the relative expression level of Beclin1 protein were increased (all P<0.05). Conclusion Autophagy occurs when inflammatory reaction occurs in the lungs of silicosis model rats; autophagy has inhibitory effect on pulmonary inflammation.

The effects of rapamycin and 3-methyladenine on nitric oxide synthase enzymes in cisplatin-induced neurotoxicity / Efectos de la rapamicina y la 3-metiladenina sobre la enzima óxido nítrico sintasa en la neurotoxicidad inducida por cisplatino

SUMMARY: The aim of this study was to determine the relationship of autophagy-enhancing rapamycin (RAPA) and autophagy- inhibitor 3-methyladenine (3-MA) with Nitric oxide synthases (NOS) in Cisplatin (CIS)-induced neurotoxicity in rats. Rats were divided into 4 groups (n=10): Control was applied saline, CIS (a single dose of 8mg/kg intraperitoneal (i.p.) on 7th day of experiment), RAPA+CIS (2 mg/kg/i.p. RAPA per day and 8 mg/kg/i.p. CIS on 7th day), 3-MA+CIS (15 mg/kg/i.p. 3-MA per day and 8 mg/kg/i.p. CIS on 7th day). Rats were sacrificed under anesthesia. Brain tissues were evaluated histopathologically. eNOS, Inos, nNOS and MAP 2 immunostaining were performed to determine the expression levels of these proteins among groups. Superoxide dismutase (SOD), Catalase (CAT), Malondialdehyde (MDA) and Interleukin IL-6 levels in brain tissue and serum nitric oxide (NO) level were measured by ELISA assay. In histopathological evaluation, neurodegeneration was seen in the CIS group. There was an increase in eNOS, iNOS and nNOS immunostaining in CIS group. While MAP2 immunostaining of the CIS group decreased. There was a decrease in SOD and CAT levels of brain tissue in CIS group. However, there was an increase in MDA, IL-6 and NO levels of brain tissue in CIS group. We found that antioxidant capacity increase while, inflammation and nitric oxide levels decreased in the RAPA-treated group. 3-MA does not have a significant effect. We suggest that CIS-induced neurotoxicity is more effective than Rapa 3-MA and may also be linked to NOS enzymes.

RESUMEN: El objetivo de este estudio fue determinar la relación de la rapamicina potenciadora de la autofagia (RAPA) y el inhibidor de la autofagia 3-metiladenina (3-MA) con óxido nítrico sintasas (NOS) en la neurotoxicidad inducida por cisplatino (CIS) en ratas. Las ratas se dividieron en 4 grupos (n = 10): grupo control se aplicó solución salina, CIS (una dosis única de 8 mg / kg intraperitoneal (ip) el día 7 del experimento), RAPA + CIS (2 mg / kg / ipRAPA por día y 8 mg / kg / ip CIS el día 7), 3-MA + CIS (15 mg / kg / ip 3-MA por día y 8 mg / kg / ip CIS el día 7). Las ratas se sacrificaron bajo anestesia y los tejidos cerebrales fueron analizados histopatológicamente. Se realizaron inmunotinciones con eNOS, Inos, nNOS y MAP 2 para determinar los niveles de expre- sión de estas proteínas entre los grupos. Se midieron los niveles de superóxido dismutasa (SOD), catalasa (CAT), malondialdehído (MDA) e interleucina IL-6 en el tejido cerebral y el nivel de óxido nítrico (NO) en suero mediante ensayo ELISA. En la evaluación histopatológica, se observó neurodegeneración en el grupo CIS. Hubo un aumento en la inmunotinción de eNOS, iNOS y nNOS en el grupo CIS. Mientras que la inmunotinción de MAP2 del grupo CIS disminuyó. Hubo una disminución en los niveles de SOD y CAT del tejido cerebral en el grupo CIS, sin embargo, hubo un aumento en los niveles de MDA, IL-6 y NO en el tejido cerebral en el grupo CIS. Observamos que la capacidad antioxidante aumentó, mientras que la inflamación y los niveles de óxido nítrico disminuyeron en el grupo tratado con RAPA. 3-MA no tiene un efecto significativo. Sugerimos que la neurotoxicidad inducida por CIS es más eficaz que Rapa 3-MA y también puede estar relacio- nada con las enzimas NOS.

Targeting autophagy using small-molecule compounds to improve potential therapy of Parkinson's disease

Parkinson's disease (PD), known as one of the most universal neurodegenerative diseases, is a serious threat to the health of the elderly. The current treatment has been demonstrated to relieve symptoms, and the discovery of new small-molecule compounds has been regarded as a promising strategy. Of note, the homeostasis of the autolysosome pathway (ALP) is closely associated with PD, and impaired autophagy may cause the death of neurons and thereby accelerating the progress of PD. Thus, pharmacological targeting autophagy with small-molecule compounds has been drawn a rising attention so far. In this review, we focus on summarizing several autophagy-associated targets, such as AMPK, mTORC1, ULK1, IMPase, LRRK2, beclin-1, TFEB, GCase, ERR

Autophagy inhibitor 3-methyladenine could not modulate rotenone neurotoxicity in primary mesencephalic cell culture / El inhibidor de la autofagia 3-metiladenina no logra modular la neurotoxicidad de la rotenona en el cultivo primario de células mesencefálicas

Dysregulated autophagy, whether excessive or downregulated, has been thought to be associated with neurodegenerative disorders including Parkinson's disease. Accordingly, the present study was carried out to investigate whether 3-methyladenine, an autophagy inhibitor, can modulate the effects of rotenone on dopaminergic neurons in primary mesencephalic cell culture. Cultures were prepared from embryonic mouse mesencephala at gestation day 14. Four groups of cultures were treated on the 10th DIV for 48 h as follows: the first was kept as an untreated control, the second was treated with 3-methyladenine alone (1, 10, 100, 200 mM), the third was treated with 20 nM rotenone and the fourth was co-treated with 20 nM rotenone and 3-methyladenine (1, 10, 100, 200 mM). On the 12th DIV, cultured cells were stained immunohistochemically against tyrosine hydroxylase and culture media were used to measure the levels of lactate dehydrogenase. 3methyladenine had no effects on both the survival of dopaminergic neurons and the release of lactate dehydrogenase. Rotenone significantly decreased the number of dopaminergic neurons and increased the levels of lactate dehydrogenase in the culture media. When cultures concomitantly treated with 3-methyladenine and rotenone, 3-methyladenine had no effect against rotenone-induced dopaminergic cell damage and lactate dehydrogenase release into the culture medium. In conclusion, the autophagy inhibitor 3-methyladenine could not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.

Se estima que la autofagia desregulada, ya sea excesiva o con baja regulación, está asociada con trastornos neurodegenerativos, incluyendo la enfermedad de Parkinson. En consecuencia, el se realizó este estudio para investigar si la 3metiladenina, un inhibidor de la autofagia,puede modular los efectos de la rotenona en las neuronas dopaminérgicas en el cultivo primario de células mesencefálicas. Los cultivos se prepararon a partir de mesencéfalo de ratón embrionario el día 14 de gestación. Cuatro grupos de cultivos se trataron en el 10º DIV durante 48 h de la siguiente manera: el primer grupo se mantuvo como un control no tratado, el segundo se trató con 3-metiladenina sola (1, 10, 100, 200 mM), el tercer grupo se trató con rotenona 20 nM y el cuarto se trató conjuntamente con rotenona 20 nM y 3-metiladenina (1, 10, 100, 200 mM). En el 12º DIV; las células cultivadas fueron tratadas mediante tinción inmunohistoquímica en tirosina hidroxilasa y se usaron medios de cultivo para medir los niveles de lactato deshidrogenasa. La 3-metiladenina no tuvo efectos tanto en la supervivencia de las neuronas dopaminérgicas como en la liberación de lactato deshidrogenasa. La rotenona disminuyó significativamente el número de neuronas dopaminérgicas y se observó un aumento de los niveles de lactato deshidrogenasa en los medios de cultivo. Cuando los cultivos tratados concomitantemente con 3-metiladenina y rotenona, la 3metiladenina no tuvo efecto contra el daño celular dopaminérgico inducido por la rotenona y la liberación de lactato deshidrogenasa en el medio de cultivo. En conclusión, el inhibidor de la autofagia 3-metiladenina no moduló el daño celular dopaminérgico inducido por la rotenona en el cultivo celular mesencefálico primario.

Cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): therapeutic and pathophysiological implications

The hepatic endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) are mixed-function oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance. P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover. Such P450 proteolytic turnover occurs through a process known as ER-associated degradation (ERAD) that involves ubiquitin-dependent proteasomal degradation (UPD) and/or autophagic-lysosomal degradation (ALD). Herein, on the basis of available literature reports and our own recent findings of as well as experimental studies, we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance. We specifically (i) describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibody pathogenesis in drug-induced acute hypersensitivity reactions and liver injury, or viral hepatitis; (ii) discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates; and (iii) detail the pathophysiological consequences of disrupted P450 ERAD, contributing to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) under certain synergistic cellular conditions.

Novel fluorescent probes of 10-hydroxyevodiamine: autophagy and apoptosis-inducing anticancer mechanisms

Natural product evodiamine and its derivatives represent a promising class of multi-target antitumor agents. However, the clinical development of these compounds has been hampered by a poor understanding of their antitumor mechanisms. To tackle this obstacle, herein, novel fluorescent probes were designed to elucidate the antitumor mode of action of 10-hydroxyevodiamine. This compound was proven to be distributed in the mitochondria and lysosomes and to act by autophagy and apoptosis mechanisms.

MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest-a phenomenon known as mitotic catastrophe, which is followed by immediate cell death apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

Hair regeneration in mice was promoted by 3-methyladenine through inhibiting autophagy flux / 中国药科大学学报

@#To explore the role of autophagy in hair follicle cycle and whether 3-methyladenine(3-MA)could promote hair regeneration in C57BL/6 mice through inhibiting autophagy flux, hair regeneration model of C57BL/6 mice was induced on the dorsal skin by depilation, and 3-MA was intraperitoneally injected to investigate hair regrowth, meanwhile vehicle and rapamycin(RAPA)were used as the controls. Results showed that 3-MA could obviously promote hair regrowth in depilated C57BL/6 mice. Furtherly, haematoxylin-eosin staining, immunohistochemical staining, immunofluorescence and Western blot tests were used to investigate the autophagy signals and the cell proliferation. Results showed that the expression of Beclin1 and LC3B II/I ratio were significantly decreased. Expression of P62 and Ki67 were increased, as well as the CD34 and CD49f double-labeled hair follicle stem cells were obviously increased inside bulge areas in 3-MA group, while contrarily in RAPA group. These results affirmed 3-MA, an autophagy inhibitor, could promote hair regeneration in depilated C57BL/6 mice by facilitating the transformation of hair follicle from telogen to anagen. 3-MA and other analogous autophagy inhibitors probably have a potential usage in future therapy in human telogen effluvium diseases.

3-Methyladenine ameliorates CCl4-induced liver fibrosis and gut microbiota in mice / 临床检验杂志

Objective@#To investigate the role of gut microbiota in the amelioration of liver fibrosis induced by CCl4 in mice by 3-methyladenine (3-MA). @*Methods@#Fifteen mice were randomly divided into normal control group, liver fibrosis group and 3-MA treatment group. The liver fibrosis model was established by injecting CCl4, and the mice in the 3-MA treatment group were injected 3-MA additionally from the third week onwards. After 8 weeks, all of the mice were sacrificed and their blood, liver tissue and fecal samples were collected to analyze serum ALT, AST, GGT levels, liver histopathology and gut microbiota. @*Results@#Compared with the liver fibrosis group, serum ALT and AST levels in 3-MA treatment group decreased obviously ([68.6±4.2] U/L vs [111.0±7.8] U/L, [179.0±12.9] U/L vs [253.2±26.7] U/L, P＜0.01), and the degree of hepatic histopathological changes was reduced. The intestinal flora in three groups were distinguished by principal coordinate analysis (PCoA) and non-metric multi-dimensional scaling (NMDS) analysis. Compared with the normal control group, there were decreased Alpha diversity of intestinal community, reduced significantly abundance of Lachnospiraceae, and increased obviously abundance of Actinobacteria and Desulfovibrionacea in the liver fibrosis group (P＜0.05). Compared with the liver fibrosis group, there were increased Alpha diversity of intestinal community, increased significantly abundance of Lachnospiraceae and Blautia, and reduced abundance of Bifidobacteriaceae in the 3-MA treatment group (P＜0.05). In addition, the abundance of Lactobacillaceae in the 3-MA treatment group was significantly higher than that in the normal control group (P＜0.05). @*Conclusion@#3-MA improves the liver fibrosis of mice induced by CCl4, and gut microbiota may play an active role in this process.

Mechanism of cell autophagy for regulating skeletal muscle wasting of rats after severe burns / 中华烧伤杂志

Objective@#To investigate the mechanism of cell autophagy for regulating skeletal muscle wasting of rats after severe burns.@*Methods@#Seventy-two Sprague-Dawley rats were collected and divided into sham injury group, simple burn group, burn+ phosphate buffer solution (PBS) group, and burn+ 3-methyladenine (3-MA) group according to the random number table, with 18 rats in each group. Rats in simple burn group, burn+ PBS group, and burn+ 3-MA group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns). Rats in sham injury group were sham injured. Immediately after burns and fluid resuscitation, rats in burn+ PBS group were intraperitoneally injected with 1 mL PBS, and rats in burn+ 3-MA group were intraperitoneally injected with 1 mL 3-MA (125 g/L). On post injury day 3 and 7, the weights of anterior tibial muscle of right hind limbs and body of rats were measured to calculate percentage of anterior tibial muscle of right hind limbs weight. Protein expressions of microtubule related protein 1 light chain 3A (LC3A) and Beclin-1 of anterior tibial muscle were observed by immunofluorescence method and detected by Western blotting, and ratio of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ was calculated. Data were processed with analysis of variance of factorial design, one-way analysis of variance, t-test and Bonferroni correction.@*Results@#On post injury day 3 and 7, percentages of anterior tibial muscle of right hind limbs weight of rats in simple burn group were (0.148±0.009)% and (0.134±0.018)%, respectively, which were significantly lower than those in sham injury group [(0.203±0.009)%, (0.181±0.015)%, t=10.585, 4.913, P<0.01]. Percentages of anterior tibial muscle of right hind limbs weight of rats in burn+ 3-MA group were (0.187±0.004)% and (0.192±0.009)%, respectively, which were obviously higher than those in burn+ PBS group [(0.162±0.005)%, (0.167±0.005)%, t=9.564, 5.948, P<0.01]. On post injury day 3 and 7, protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group, while protein expressions of Beclin-1 and microtubule related protein 1 LC3A of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group. Ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in simple burn group were significantly higher than those in sham injury group (t=3.461, 3.353, P<0.05), while ratios of microtubule related protein 1 LC3A-Ⅱ to LC3A-Ⅰ of anterior tibial muscle of rats in burn+ 3-MA group were significantly lower than those in burn+ PBS group (t=3.129, 3.977, P<0.05).@*Conclusions@#Cell autophagy induced by severe burns is involved in the process of skeletal muscle wasting of rats, and inhibition of cell autophagy may contribute to the remission of skeletal muscle wasting of rats induced by burns.

Effect of 3-methyladenine on autophagy, migration and invasion in epithelial ovarian cancer cells under hypoxia / 上海交通大学学报（医学版）

Objective: To investigate the effect of 3-methyladenine (3-MA) on autophagy, migration and invasion of epithelial ovarian cancer (EOC) cells under hypoxia. Methods: Different concentrations of 3-MA were used to treat EOC cells in hypoxic environment. The expression of autophagy-associated protein LC3 was detected by Western blotting. The autophagosome formation was observed by transmission electron microscopy. The proliferation, adhesion, migration and invasion of EOC cells were determined by thiazole blue colorimetry, adhesion test, Transwell migration test and Matrigel invasion test, respectively. Results: 3-MA was able to inhibit the increase of LC3- Ⅱ and autophagosome formation induced by hypoxia. In hypoxic environment, the survival rate of EOC cells was significantly decreased by 5.00 mmol/L 3-MA for 24 h (P=0.000). The adhesion ability of EOC cells was significantly decreased by 2.50 mmol/L 3-MA for 6 h (P=0.007). 1.25 mmol/L and 2.50 mmol/L 3-MA could inhibit the migration and invasion of EOC cells in hypoxic environment (P<0.01). Conclusion: 3-MA can inhibit the autophagy, migration and invasion of EOC cells in hypoxic environment.

Effects of Remifentanil Preconditioning Attenuating Oxidative Stress in Human Dermal Fibroblast

Human dermal fibroblast is essential in wound healing of the skin through the synthesis of extracellular matrix proteins. With respect to oxidative stress, the effects of remifentanil on human dermal fibroblast have received little attention. Therefore, we investigated the effects of remifentanil on the apoptosis and autophagic reaction of human dermal fibroblasts under oxidative stress. The subjects were divided into the following groups: Control group: cells were incubated at 37℃ in a humidified atmosphere with 5% CO₂. Hydrogen peroxide (H₂O₂) group: cells were exposed to H₂O₂ for 2 h. RPC/H₂O₂ group: cells were pretreated with remifentanil for 2 h and exposed H₂O₂ for 2 h. 3-MA/RPC/H₂O₂ group: cells were pretreated with 3-methyladenine (3-MA) and remifentanil for 1 h and 2 h, respectively. We measured cell viability using MTT assay. Western blot analysis was used to determine the expression levels of proteins associated with apoptosis and autophagy. Quantification of apoptotic cells was performed using flow cytometer analysis, and autophagic vacuoles were observed under a fluorescence microscope. Remifentanil treatment increased the proliferation of human dermal fibroblast and decreased apoptotic cell death, enhancing autophagic activity under oxidative stress. However, 3-MA, the autophagy pathway inhibitor, inhibited the protective effect of remifentanil in oxidative stress. This study demonstrates that remifentanil activated autophagy and decreased apoptotic death of human dermal fibroblasts under oxidative stress. Our results suggest that remifentanil may help in the treatment of oxidative stress.

3-MA combined with TSA inhibits growth of triple-negative breast cancer cells / 中国病理生理杂志

AIM: To investigate the effect of 3-methyladenine (3-MA) combined with trichostatin A (TSA) on triple-negative breast cancer cells and the mechanism involved.METHODS: The viability of MDA-MB-231 cells was detected by MTT assay, the migration ability was determined by scratch assay, and the expression of autophagy-related proteins was detected by Western blot.RESULTS: TSA significantly inhibited the viability of MDA-MB-231 cells in a time-and dose-dependent manner.The results of scratch assay showed that TSA inhibited cell migration ability.Western blot data indicated that TSA resulted in a moderate increase in LC3-Ⅱ expression.Moreover, 3-MA inhibited cell autophagy induced by TSA, and combination of 3-MA and TSA further inhibited the viability of MDA-MB-231 cells.CONCLUSION: Combination of 3-MA and TSA may effectively inhibit the growth of triple-negative breast cancer cells.

Protective Effects of 3-Methyladenine against Lipopolysaccharide-induced Acute Lung Injury in Mice and the Mechanism Study / 中国药房

OBJECTIVE:To study the protective effects of autophagy inhibitor 3-Methyladenine (3-MA) against lipopolysac-charide(LPS)-induced acute lung injury in mice and its mechanism. METHODS:Mice were randomly divided into normal control group,model group (LPS 15 mg/kg),drug control group (3-MA 20 mg/kg),low-dose and high-dose groups (LPS 15 mg/kg+3-MA 20,40 mg/kg),with 10 mice in each group. Except for normal control group and drug control group,other groups were giv-en LPS intraperitoneally to induce acute lung injury model,and drug control group and low-dose and high-dose groups were given equivalent dose of 3-MA intraperitoneally 1 h before modeling. 6 h after modeling,lung wet/drug mass ratio (W/D) was deter-mined respectively,and pathology change of lung tissue was observed by HE staining. TNF-α,NF-κB p65,LC3BⅡ/Ⅰ and Cleaved-caspase-3 protein expression were detected by Western blot. RESULTS:Compared with normal control group,W/D, TNF-α,NF-κB p65,LC3BⅡ/Ⅰ and Cleaved-caspase-3 protein expression increased in model group (P<0.01). Compared with model group,W/D,the expression of TNF-α,NF-κB p65,LC3BⅡ/Ⅰ and Cleaved-caspase-3 protein decreased in low-dose group (P<0.05),white just only LC3BⅡ/Ⅰ protein decreased high-dose group(P<0.01). CONCLUSIONS:In LPS-induced acute lung injury model in mice,the excessive autophagy could activate the NF-κB pathway and involve the inflammatory responses and induce lung cells apoptosis. The moderate autophagy inhibition by 3-MA can ameliorate inflammatory response and protect lung tissue.

Effect of autophagy on necroptosis of renal tubular epithelial cells in sub-total nephrectomy rats / 中国病理生理杂志

[ ABSTRACT] AIM: To explore whether autophagy is involved in the excessive death of renal tubular epithelial cells in subtotal nephrectomy ( SNx) rats and the relationship between autophagy and necroptosis in the kidney of SNx rats. METHODS:Male Sprague-Dawley rats were randomly assigned to control group ( n=6 ) and SNx group ( n=42 ) .The rats in SNx group were subjected to SNx.Sham surgery was performed in the rats in control group.The rats in SNx group were divided into subgroups at 0, 4, 8 and 12 weeks ( n=6) and the other rats in SNx group were divided into SNx+vehi-cle group, SNx+necrostatin-1 (Nec-1) group and SNx+3-methyladenine (3-MA) group.The expression of RIP1, RIP3, LC3 and beclin-1 at mRNA and protein levels was measured at 0, 4, 8 and 12 weeks by qPCR and immunohistochemistry. The effects of Nec-1 or 3-MA on the protein expression of LC3-I, LC3-II and beclin-1, and production of reactive oxygen species ( ROS) in the rat kidney were determined by Western blot and DCFH-DA staining.The death of renal tubular epi-thelial cells in the SNx rats was observed by TUNEL staining and electron microscopy.Finally, the effects of Nec-1 and 3-MA on blood urea nitrogen ( BUN) , serum creatinine ( SCr) and the pathological changes of the renal tissues were ana-lyzed.RESULTS:The highest mRNA and protein levels of RIP1, RIP3, LC3 and beclin-1 appeared at the 8th week after SNx (P cells were decreased in the SNx rats treated with Nec-1 and 3-MA (P<0.01), but 3-MA did not reduce the increased con-centration of ROS.In addition, treatment with Nec-1 and 3-MA obviously reduced BUN, SCr (P<0.05), glomeruloscle-rosis index and tubulointerstitial injury score (P<0.01).CONCLUSION:Autophagy participates in the excessive death of renal tubular epithelial cells in SNx rats.Inhibition of autograph prevents necroptotic cell death of renal tubular cells, and alleviates chronic renal injury in SNx rats.

Effect of 3-MA on camptothecin-induced cervical cancer Hela cell apoptosis / 中国免疫学杂志

Objective:To explore the effect of autophagy inhibitor 3-methyladenine(3-MA) on camptothecin(CPT)-induced Hela cell apoptosis.Methods:MTT assays were carried out to determine the optimal concentration and time of CPT on Hela cells and the effect of different drugs on Hela cell proliferation activity .After Hela cells were treated with different drugs ,the changes of autophagy marker protein( microtubule-associated protein 1 light chain 3,LC3),p62 and apoptosis-related protein were detected using Western blot and immunofluorescence ( IF) .DAPI ( nuclear ) staining was used to observe cell apoptosis rate .Results: In CPC-treated Hela cells,Hela cell proliferation activity declined dramatically ,and autophagy could be induced to occur .Compared with CPT group ,the cell proliferation activity was lower in CPT combined with 3-MA group,the level of autophagy decreased ,but the apoptosis rate significantly increased.Conclusion:CPT can induce autophagy while inducing Hela cell death .Hela cells chemosensitivity to CPT treatment can be enhanced by 3-MA inhibiting autophagy .

Influence of antophagy in radiation sensitivities of oral squamous cell carcinoma CAL-27 and KB cells and its mechanisms / 吉林大学学报(医学版)

Objective: To use autophagy inhibitors combined with radiation to treat the oral squamous cell carcinoma CAL-27 and KB cells,and to explore the influence of autophagy in the oral cancer radiation sensitivity and its mechanisms. Methods:The human oral squamous cell carcinoma CAL-27 and KB cells were divided into control group,CQ group,3-MA group,IR group,CQ+IR group,and 3-MA+IR group. The survival rate was detected by MTT method and the autophagy of CAL-27 cells was observed by immunofluorescence method and laser scanning confocal microscope.The expression levels of LC3 and beclin-1 were detected by Western blotting method. The apoptotic rate was determined by flow cytometry with Annexin Ⅴ/PI doulde staining. Results:Compared with IR group,the survival rates in 3-MA + IR and CQ+ IR groups were signifcantly decreased (P < 0.05 ).The autophagy levels of cells in IR group were significantly higher than those in control group, CQ group, 3-MA group,CQ+IR group,and 3-MA+IR group (P <0.05).The expression levels of LC3 and beclin-1 proteins in IR group were significantly higher than those in control group,CQ+ IR group,and 3-MA+ IR group (P < 0.05). The apoptotic rates in IR,3-MA+ IR,and CQ+ IR groups were markedly higher than those in control group. Compared with IR group,the apoptotic rates in CQ+IR and 3-MA+IR groups were significantly increased (P <0.05).Conclusion:Radiotherapy can induce the increase of autophagy level of oral squamous cell carcinoma cells. Inhibiors of autophagy can increase the radio-sensitivity of oral squamous cell carcinoma cells by inhibiting the proliferation and inducing the apoptosis.